Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly(ethylenimine).

A target-specific delivery system of green fluorescent protein (GFP) small interfering RNA (siRNA) plasmid DNA was developed by using folate-modified cationic polyethylenimine (PEI). A GFP siRNA plasmid vector (pSUPER-siGFP), which inhibits the synthesis of GFP, was constructed and used for suppressing GFP expression in folate receptor over-expressing cells (KB cells) in a target-specific manner. A PEI-poly(ethylene glycol)-folate (PEI-PEG-FOL) conjugate was synthesized as a pSUPER-siGFP plasmid gene carrier. KB cells expressing GFP were treated with various formulations of pSUPER-siGFP/PEI-PEG-FOL complexes to inhibit expression of GFP. The formulated complexes were characterized under various conditions. Their GFP gene inhibition and cellular uptake behaviors were explored by confocal microscopy and flow cytometry analysis. pSUPER-siGFP/PEI-PEG-FOL complexes inhibited GFP expression of KB cells more effectively than pSUPER-siGFP/PEI complexes with no folate moieties and showed far reduced extent of inhibition for folate receptor deficient cells (A549 cells). The results indicated that folate receptor-mediated endocytosis was a major pathway in the process of cellular uptake, suggesting that targeted delivery of siRNA vector could be achieved to a specific cell.     

Folate receptor targeted biodegradable polymeric doxorubicin micelles.

Biodegradable polymeric micelles, self-assembled from a di-block copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG), were prepared to achieve folate receptor targeted delivery of doxorubicin (DOX). In the di-block copolymer structure of PLGA-b-PEG, DOX was chemically conjugated to a terminal end of PLGA to produce DOX-PLGA-mPEG, and folate was separately conjugated to a terminal end of PEG to produce PLGA-PEG-FOL. The two di-block copolymers with different functional moieties at their chains ends were physically mixed with free base DOX in an aqueous solution to form mixed micelles. It was expected that folate moieties were exposed on the micellar surface, while DOX was physically and chemically entrapped in the core of micelles. Flow cytometry and confocal image analysis revealed that folate conjugated mixed micelles exhibited far greater extent of cellular uptake than folate unconjugated micelles against KB cells over-expressing folate receptors on the surface. They also showed higher cytotoxicity than DOX, suggesting that folate receptor medicated endocytosis of the micelles played an important role in transporting an increased amount of DOX within cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that when systemically administered, tumor volume was significantly regressed. Biodistribution studies also indicated that an increased amount of DOX was accumulated in the tumor tissue.     

Folate receptor-mediated intracellular delivery of recombinant caspase-3 for inducing apoptosis.

Recombinant reversed caspase-3 (rev-caspase-3) is a pro-apoptotic gene capable of intracellular autocatalytic processing, which leads to programmed cell death. Folate receptor-specific intracellular delivery of the rev-caspase-3 gene into KB cells over-expressing folate receptors was explored by employing the folate-poly(ethylene glycol)-polyethylenimine (FOL-PEG-PEI) conjugate as a nonviral polymeric carrier. Using luciferase as a reporter gene, the conditions for formulation of DNA/polymer polyplexes were pre-optimized to attain the highest folate receptor-mediated gene transfection efficiency. FOL-PEG-PEI conjugate complexed with rev-caspase-3 plasmid in an optimized condition gave rise to a great increase in expression and activation of exogenous rev-caspase-3 in KB cells when pretreated with doxorubicin. The synthesized conjugate exhibited higher transfection efficiency than other commercially available transfection agents due to a unique mechanism of folate-receptor mediated endocytic gene transfer. The transfected cells showed a significant extent of apoptosis by rev-caspase-3. This study suggests the potential of using folate-receptor-mediated delivery of rev-caspase-3 gene for inducing tumor cell death in a target-specific manner.     

Retargeting of viral vectors to the folate receptor endocytic pathway.

Viral vectors with high transfection efficiencies are not always those with optimal target cell binding specificities. As a consequence, virus pseudotyping has been developed to endow transfection competent viruses with improved cell binding specificities and affinities. We have hypothesized that chemical conjugation of a virus to a cell specific ligand might also alter its target cell specificity and produce a virus that would transfect only the desired cell type. To test this concept, an ecotropic replication-defective myeloproliferative sarcoma retrovirus and an amphotropic murine adenovirus containing the gene for beta-galactosidase were chemically derivatized with folic acid. As expected from its strong ecotropism, the unmodified retrovirus did not induce beta-galactosidase expression in nonhost KB cells, while the amphotropic adenovirus yielded high levels of gene expression in the same cell line. Surprisingly, although folate derivatization enabled avid binding of both viruses to folate receptor expressing KB cells, the folate conjugation did not promote retroviral gene expression and actually prevented the normal beta-galactosidase expression seen with the adenoviral vector. The fact that co-administration of excess free folic acid to block uptake by folate receptor-mediated endocytosis restored adenoviral gene expression to the level obtained with unmodified virus suggests that folate derivatization per se does not hamper viral activity. We, therefore, conclude that neither retroviral nor adenoviral delivery via the folate endocytosis pathway is compatible with viral gene expression in KB cells.     

A dual-ligand approach for enhancing targeting selectivity of therapeutic nanocarriers.

Conjugation of ligands to nano-scale drug carriers targeting over-expressed cell surface receptors is a promising approach for delivery of therapeutic agents to tumor cells. However, most commonly utilized ligands are directed at receptors expressed not only on target cells but also on other cells in the body, leading to unintended uptake in these off-target cells. In this study, a novel, dual-ligand approach is reported, which targets tumor cells while sparing off-target cells by exploiting the fact that tumor cells typically over-express multiple types of surface receptors. This approach was tested in the human KB cell line, which over-expresses both folate receptor (FR) and the epidermal growth factor receptor (EGFR). Liposomal nanocarriers loaded with doxorubicin and bearing controlled numbers of both folic acid and a monoclonal antibody against the EGFR were designed. Cytotoxicity was used to determine targeting selectivity of the designed carriers in vitro by utilizing KB cells expressing both FR and EGFR and off-target control cells in which one or both receptors were blocked. The data demonstrates that nanocarriers can be designed to achieve toxicity only when all targeted receptors are available, providing an approach to improve selectivity over current single-ligand approaches.     

Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate.

For folate-receptor-targeted anti-cancer therapy, doxorubicin aggregates in a nano-scale size were produced employing doxorubicin-polyethylene glycol-folate (DOX-PEG-FOL) conjugate. Doxorubicin and folate were respectively conjugated to alpha- and omega-terminal end group of a PEG chain. The conjugates assisted to form doxorubicin nano-aggregates with an average size of 200 nm in diameter when combined with an excess amount of deprotonated doxorubicin in an aqueous phase. Hydrophobically deprotonated doxorubicin molecules were aggregated within the core, while the DOX-PEG-FOL conjugates stabilized the aggregates with exposing folate moieties on the surface. The doxorubicin nano-aggregates showed a greater extent of intracellular uptake against folate-receptor-positive cancer cells than folate-receptor-negative cells, indicating that the cellular uptake occurred via a folate-receptor-mediated endocytosis mechanism. They also exhibited more potent cytotoxic effect on KB cells than free doxorubicin. In a human tumor xenograft nude mouse model, folate-targeted doxorubicin nano-aggregates significantly reduced the tumor volume compared to non-targeted doxorubicin aggregates or free doxorubicin. These results suggested that folate-targeted doxorubicin nano-aggregates could be a potentially useful delivery system for folate-receptor-positive cancer cells.     

Controlled targeting of liposomal doxorubicin via the folate receptor in vitro.

Differential expression of folate receptor has been exploited to target liposomes to tumors. Astrogliomas express low folate receptor levels and are typically surrounded by normal cells expressing little or no folate receptors. While targeting cells with high over-expression of folate receptor (KB and HeLa) has been demonstrated, it is unclear whether targeting tumors expressing low levels of folate receptor is possible. In this study, it was demonstrated that optimizing the number of targeting ligands (folic acid) enables differential liposomal doxorubicin uptake in C6 glioma while sparing healthy cortical cells. By micellization of folate conjugates and their controlled insertion into pre-formed liposomes, tight control over the number of targeting ligands per liposome was demonstrated. Doxorubicin uptake in KB and C6 cells was dependent on the number of targeting ligands, while cortical cells showed increasing non-specific uptake with ligand number. Co-culture of C6 glioma with cortical cells confirmed preferential uptake in C6 glioma relative to cortical cells. A cell kill experiment showed that folate-targeted liposomal doxorubicin is cytotoxic and slows proliferation of KB and C6 cells with minimal effect on cortical cells. Therefore modulation of targeting ligand number enables significant differential uptake of doxorubicin in cells with low levels of folate receptor.     

Transport of macrophage Fc receptors and Fc receptor-bound ligands to lysosomes

Mouse macrophage Fc receptors specific for IgG1/IgG2b mediate the binding and pinocytic uptake of soluble IgG-containing antibody-antigen complexes. Internalization of these multivalent IgG complexes is accompanied not only by the intracellular degradation of the ligand, but also by a net decrease in the number of plasma membrane Fc receptors and an accelerated rate of receptor turnover. In contrast, internalized receptors bound to a monovalent ligand, the high affinity Fab fragment of the antireceptor mAb 2.4G2, escape degradation by rapidly recycling to the cell surface. In this paper, we have characterized the intracellular pathway involved in the endocytosis and transport of Fc receptors in the J774 macrophage cell line. The results show that the uptake of multivalent ligands follows the normal pathway of receptor-mediated endocytosis: internalization in clathrin-coated pits and coated vesicles, delivery to endosomes, and finally to acid hydrolase-rich lysosomes. Immunoprecipitation of radiolabeled receptor from Percoll density gradients showed that endocytosis of the IgG complexes also results in the concomitant transport of the receptor to lysosomes. Although uptake of the monovalent Fab fragment had no detectable effect on intracellular receptor distribution, preparations of 2.4G2 Fab rendered multivalent by adsorption to colloidal gold were as effective as the IgG complexes at causing lysosomal accumulation of internalized receptors. Thus, it is likely that the down-regulation and degradation of Fc receptors which occurs during the endocytosis of antibody-antigen complexes is due to the transport of internalized receptors to lysosomes. Moreover, the ability of certain Fc receptor-bound ligands to interfere with receptor recycling and trigger lysosomal transport seems to depend on ligand valency rather than on the presence or absence of Fc domains on intact IgG molecules.     

Enhanced in vitro DNA transfection efficiency by novel folate-linked nanoparticles in human prostate cancer and oral cancer.

Novel folate-linked, cationic nanoparticles (NPs) were developed and evaluated for potential use for gene delivery to human oral cancer (KB cells) and human prostate cancer (LNCaP cells), which abundantly expressed folate binding proteins. Folate-polyethylenglycol-distearoylphosphatidylethanolamine conjugate (f-PEG-DSPE) was incorporated in NPs composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and Tween 80. NP-0.3FT, -1FT and -1FLT, which contain 0.3 and 1 mol% f-PEG2000-DSPE, and 1 mol% f-PEG5000-DSPE, respectively, showed about 100-200 nm in size. The NP/plasmid DNA complex (nanoplex) remained in an injectable size (230-340 nm) and slightly increased its size in serum. The association of NP-1FT with KB cells was enhanced by f-PEG2000-DSPE and was blocked by co-incubation with free folic acid in medium. In transfection activity, the NP-1FT, but not NP-1FLT, showed high activity into KB and LNCaP cells in the presence of serum. The NP-0.3FT also showed high activity into LNCaP cells, but not KB cells. In RT-PCR analysis, KB cells strongly expressed folate receptors mRNA, but LNCaP cells did not. In contrast, LNCaP cells expressed mRNA of prostate-specific membrane antigen (PSMA), which interacts with the folate substrate. Uptake mechanism of folate-linked NPs in LNCaP cells may be different from that in KB cells. This is the first report that folate-linked NPs selectively deliver the DNA to LNCaP cells, suggesting that such NPs are potentially targeted vectors to prostate cancer for gene delivery.     

A receptor-mediated gene delivery system using streptavidin and biotin-derivatized, pegylated epidermal growth factor.

An efficient receptor-mediated non-viral gene delivery formulation based on mono-pegylated recombinant human epidermal growth factor (EGF) was developed using a streptavidin-biotin system. Biotin-derivatized and mono-pegylated EGF was prepared by conjugating a biotin-PEG-NHS derivative to EGF and purified through a chromatographic method. Luciferase plasmid DNA and polyethylenimine (PEI) were complexed to form positively charged nanoparticles on which negatively charged streptavidin was first coated and then biotin-PEG-EGF conjugate was immobilized via streptavidin-biotin interaction. The EGF-PEG-biotin-streptavidin-PEI-DNA complexes were characterized in terms of their effective diameter and surface zeta (zeta)-potential value under various formulation conditions. The formulated complexes exhibited high transfection efficiency (approximately 10(8) in luciferase activity) with no inter-particle aggregation. This was attributed to enhanced cellular uptake of the resultant complexes via receptor-mediated endocytosis. Furthermore, in the presence of serum proteins, a slight decrease in transfection efficiency was observed due to the presence of PEG chains on the surface.     

Targeted delivery of antisense oligodeoxynucleotides to folate receptor-overexpressing tumor cells.

A major problem in exploring the full potential of antisense ODN is the lack of a safe and efficient delivery system. In this study a new method has been developed that is highly efficient in encapsulating ODN inside folate receptor (FR)-targeted lipid vesicles. ODN formulated in these vesicles were efficiently protected from degradation by nucleases compared to free ODN. Folate efficiently mediated intracellular delivery of ODN to KB tumor cells that overexpress FR. Delivery of EGFR antisense ODN via FR-targeted lipid vesicles resulted in a significant down-regulation of EGFR expression in KB cells and cell growth inhibition, far more efficient than that with free ODN or ODN encapsulated in ligand-free lipid vesicles. Intracellular delivery of EGFR antisense ODN also sensitized KB cells to doxorubicin (DOX) treatment. Thus targeted delivery of ODN via this novel lipid vector may have potential in treating tumors that overexpress FR.     

Induction of cancer cell-specific apoptosis by folate-labeled cationic liposomes.

We have previously reported that cationic liposomes themselves can induce apoptosis in macrophages and lymphocytes. In this paper, we attempted the cancer cell-specific delivery of cationic liposomes and the induction of apoptosis utilizing this characteristic. Cationic liposomes composed of stearylamine (SA-liposomes) induced apoptosis in human nasopharyngeal epidermoid carcinoma cells (KB cells) overexpressing the folate receptor and human fibroblasts (WI-38 cells) with no folate receptor, without showing selectivity. To recruit liposomes to cancer cells and induce apoptosis, we focused on the folate receptor and prepared folic acid-labeled liposomes using polyethyleneglycol (PEG) (folate-PEG-liposomes). Folate-PEG-liposomes showed selectivity and induced apoptosis in KB cells, but not WI-38 cells. The apoptosis occurred in a dose-dependent manner. Furthermore, folate-PEG-liposomes appear to associate with KB cells via the folate receptor, whereas SA-liposomes may associate with cells through electrostatic interactions. To confirm the contribution of the folate receptor to apoptosis of KB cells induced by folate-PEG-liposomes, the effect of folic acid on the apoptosis was examined. The addition of free folic acid drastically suppressed the apoptosis of KB cells and the percentage of cells with hypodiploid nuclei returned to the control level. Taken together, cationic liposomes labeled with folate bound to KB cells via folate receptors and, interestingly, the cationic liposomes themselves could cause apoptosis in cancer cells.     

Tumor-targeted bioconjugate based delivery of camptothecin: design, synthesis and in vitro evaluation.

Camptothecin (CPT) presents numerous challenges associated with optimal transport and delivery including variability in clinically observed effects, low target tissue concentrations and severe and unpredictable toxicity. The objective of the present study was to optimize the delivery of CPT by targeting it to cancer cells using an endogenous receptor system. A novel CPT bioconjugate was synthesized using carbodiimide chemistry with a linear poly(ethylene glycol) (PEG) and amino acid glycine as the spacer and linker respectively. Folic acid was used as the targeting ligand to take advantage of folate receptor mediated endocytosis. The bioconjugate was extensively characterized using MALDI, proton NMR, FT-IR and amino acid analysis. Furthermore, the bioconjugate was evaluated in vitro for specific targeting to folate receptor-expressing KB cells, a human nasopharyngeal carcinoma. Finally, the delivery system was evaluated for cytotoxicity using a MTT based assay. The results indicate significantly higher efficacy of the bioconjugate in comparison to CPT. A control conjugate without PEG demonstrated no improvement in efficacy over untargeted CPT emphasizing the importance of spacer between the anticancer compounds and targeting moiety. This bioconjugate represents the 'first-in-series' of targeted bioconjugates and serves as prototype for improving tumor cell concentration and efficacy.     

Effect of perturbation of specific folate receptors during in vitro erythropoiesis.

Although antisera to specific placental folate receptors inhibits the uptake of 5-methyltetrahydrofolate into cultured malignant human cells, little is known of the functional significance of folate receptors in normal human cells. Human bone marrow cells were therefore assayed for erythropoietic burst-forming units in the presence of an antihuman placental folate receptor serum and preimmune serum to determine the role of such a receptor in erythroid differentiation. When marrow cells were assayed in the presence of anti-receptor antiserum, there was (i) a threefold increase in erythropoietic burst formation and a twofold increase in the number of cells per erythroid burst; (ii) morphological evidence for nuclear/cytoplasmic dissociation of orthochromatic normoblasts composing erythroid bursts (megaloblastic erythropoiesis); (iii) intracellular folate deficiency with a 70% reduction of intracellular folate in antiserum treated cells as compared with control cells; and (iv) complete reversal of antiserum-induced changes on preincubation of antiserum with purified human placental folate receptor. These data support the conclusion that folate receptors on marrow cells provide an important function in the cellular uptake of folates during in vitro erythropoiesis. This process of folate uptake also appears to play a pivotal role in the differentiation and proliferation of erythroid progenitor cells.     

Preparation and evaluation of paclitaxel-loaded PEGylated immunoliposome

A sterically stabilized paclitaxel-loaded liposome tailored to target human breast cancer cells was developed, thereby promoting the efficiency of intracellular delivery of paclitaxel through receptor-mediated endocytosis. Results indicated that the targeting moiety (thiolated Herceptin) was successfully coupled to the distal reactive maleimide terminus of the poly (ethylene glycol)-phospholipid conjugate as well as being incorporated in the liposomal bilayers. The particle size of the PEGylated immunoliposome was maintained at about 200 nm. Confocal microscopy studies showed that the PEGylated immunoliposome was uptaken into the interior of the tumor cell through the receptor-mediated endocytosis pathway. The PEGylated immunoliposome showed substantially higher cellular uptake than the PEGylated liposome in cancer cells (BT-474 and SK-BR-3) over-expressing human epidermal growth factor receptor 2 (HER2) at 37 °C, while no difference was found in low HER2 expressing cells (MDA-MB-231) nor at low temperature (4 °C). Pharmacokinetics of paclitaxel in the PEGylated immunoliposome was compared with that in Taxol® and in the PEGylated liposome in rats. The circulating time of paclitaxel in the PEGylated immunoliposome was prolonged compared to Taxol® while slightly shortened than that in the PEGylated liposome. Therefore, the paclitaxel-loaded PEGylated immunoliposome using Herceptin could serve as a promising model for future tumor specific cancer therapy of HER2 over-expressing breast cancers.     

Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer.

Biodegradable polymeric micelles containing doxorubicin in the core region were prepared from a di-block copolymer composed of doxorubicin-conjugated poly(DL-lactic-co-glycolic acid) (PLGA) and polyethyleneglycol (PEG). The di-block copolymer of PLGA-PEG was first synthesized and the primary amino group of doxorubicin was then conjugated to the terminal hydroxyl group of PLGA, which had been pre-activated using p-nitrophenyl chloroformate. The resulting polymeric micelles in aqueous solution were characterized by measurement of size, drug loading, and critical micelle concentration. The micelles containing chemically-conjugated doxorubicin exhibited a more sustained release profile than PEG-PLGA micelles containing physically-entrapped doxorubicin. The cytotoxic activity of the micelles against HepG2 cells was greater than free doxorubicin, suggesting that the micelles containing conjugated doxorubicin were more effectively taken up cellularly, by an endocytosis mechanism rather than by passive diffusion. Confocal microscopic observation and flow cytometry analysis supported the enhanced cellular uptake of the micelles.     

Preclinical pharmacokinetics, tissue distribution, and antitumor activity of a folate-hapten conjugate-targeted immunotherapy in hapten-immunized mice

Folic acid (pteroylglutamic acid) represents a useful ligand for targeted cancer therapies because it binds to a common epithelial tumor antigen known as the folate receptor. We previously devised an immunotherapy strategy that uses a bispecific ligand, a folate-hapten (FITC) conjugate, to redirect endogenously induced anti-FITC antibodies to folate receptor-positive tumor cells following parenteral administration. Here, we present results from preclinical pharmacokinetic and tissue biodistribution studies using a radioactive folate-FITC conjugate and results from dose optimization studies done in tumor-bearing animals. Folate-FITC was found to be rapidly eliminated in non-immunized mice; however, in immunized hosts, folate-FITC was shown to form immune complexes with FITC-specific antibodies, the consequence of which was a approximately 173-fold increase in drug exposure (i.e., area under the curve). Using a newly developed ELISA assay, the extent of circulating anti-FITC antibodies occupied by parenterally given folate-FITC was determined to be proportional to the given dose. Furthermore, high doses of folate-FITC were found to promote the cosaturation of tumor cell surface folate receptors and circulating FITC-specific antibodies, blocking the immune recognition of tumor cells and thereby reducing antitumor activity. Nonetheless, by extending the duration of treatment and administering subsaturating doses of folate-FITC, enhanced antitumor response was observed in mice bearing established folate receptor-positive M109 tumors. Overall, results from the present study may help to guide clinicians through on-going clinical investigations of folate-targeted immunotherapy.     

Efficient gene delivery into human dendritic cells by adenovirus polyethylenimine and mannose polyethylenimine transfection

Gene-modified human dendritic cells (DCs) were generated by transfection with adenovirus polyethylenimine DNA (Ad/PEI/DNA) and mannose polyethylenimine DNA (ManPEI/DNA) complexes. Ad/PEI/DNA complexes have plasmid DNA bound to adenovirus particles by PEI and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yield high transduction levels and sustained expression of luciferase and green fluorescent protein reporter genes and were almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor-mediated endocytosis via mannose receptor, which is highly expressed on DCs. While gene delivery by ManPEI/DNA complexes was less efficient than by Ad/PEI transfection, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhanced transduction efficiencies and transgene expression. We also demonstrate that Ad/PEI-transfected DCs are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions, and in activating T cells from T cell receptor (TCR)-transgenic mice in an antigen-specific manner. Thus, the present study establishes the following relative order of transduction efficiencies of viral and nonviral gene delivery systems for primary human DCs: recombinant adenovirus > Ad/PEI = Ad/ManPEI > ManPEI > PEI. Ad/PEI and ManPEI transfection modes represent particularly versatile transduction systems for DCs, with ManPEI being built up exclusively of synthetic compounds.     

Dexamethasone-mediated up-regulation of the mannose receptor improves the delivery of recombinant glucocerebrosidase to Gaucher macrophages

Enzyme replacement therapy for Gaucher disease uses a recombinant glucocerebrosidase (Cerezyme) whose oligosaccharide chains have been remodeled to expose the core mannose residues. This modification promotes the uptake of the hydrolase by Gaucher-affected macrophages via mannose receptor-mediated endocytosis. However, studies revealed that amounts of the infused enzyme were also delivered to other mannose receptor-bearing cells such as the liver sinusoidal endothelial cells. To maximize the delivery of Cerezyme to macrophages, agents that increased the cell surface levels of the mannose receptor specifically on macrophages were examined. Treatment with dexamethasone improved the in vitro uptake of Cerezyme by a macrophage but not by liver sinusoidal endothelial or hepatocyte cell lines. The enhanced uptake by the macrophages was due to an increase in surface mannose receptors because the activity could be blocked by the addition of mannans. Pretreatment of rats with the glucocorticoid also preferentially enhanced the delivery of Cerezyme to the Kupffer cells and splenic macrophages. This effect of dexamethasone also applied to substrate-laden macrophages isolated from Niemann-Pick A mice. Together, these data suggest that pretreatment with dexamethasone could specifically enhance the presentation of mannose receptors on Gaucher macrophages with resultant improvement in delivery of the enzyme to the affected cells.     

Antisense oligonucleotides targeted to the human alpha folate receptor inhibit breast cancer cell growth and sensitize the cells to doxorubicin treatment

Folates are essential for cell survival and are required for numerous biochemical processes. The human alpha isoform folate receptor (alphahFR) has a very high affinity for folic acid and is considered an essential component in the cellular accumulation of folates and folate analogues used in chemotherapy. The expression of alphahFR is not detected inmost normal tissues. In contrast, high levels of the expression of alphahFR have been reported in a variety of cancer cells. The significance of alphahFR overexpression in malignant tissues has not been elucidated, but it is possible that it promotes cell proliferation not only by mediating folate uptake but also by generating other regulatory signals. The purpose of the present study was to evaluate alphahFR as a potential target for the treatment of breast cancer. Initial studies were done in nasopharyngeal carcinoma (KB) cells, which express high levels of alphahFR. In KB cells, antisense oligodeoxyribonucleotides (ODN) complementary to the alphahFR gene sequences were found to reduce newly synthesized alphahFR protein up to 60%. To examine the effect of alphahFR antisense ODNs in a panel of cultured human breast cancer cell lines, we used a tumor cell-targeted, transferrin-liposome-mediated delivery system. The data show that alphahFR antisense ODNs induced a dose-dependent decrease in cell survival. Finally, we determined that alphahFR antisense ODNs sensitized MDA-MB-435 breast cancer cells by 5-fold to treatment with doxorubicin. The data support the application of alphahFR antisense ODNs as a potential anticancer agent in combination with doxorubicin.